Defective regulation of cell secretion underlies the clinical manifestations of a number of important human diseases ranging from cystic fibrosis to secretory diarrhea and brain edema.
The regulation of metazoic cells' dimensions and internal milieu is a very high priority matter for their normal function, multiplication and survival. The cytoskeleton, formed by the fairly static microtubules and intermediate filaments, and the dynamic actin filament network act as sensors of cells' shape, dimensions, three dimensional shape and mechanical load. Excessive inflow of water and ions increase a cell's dimensions, e.g., as occurring if the cell is exposed to a hypotonic extracellular environment. In contrast, a hypertonic extracellular milieu renders the cell to shrink. Any divergence from the normal status of a cell is immediately and forcefully counteracted as a cell, for the sake of its survival and optimal function, is giving its highest priority to maintain a “normal” status. Thereby, the actin filaments and associated myosin 1 form a sensing system alarming on any divergence from the normal conditions. The actin filaments are attached to lipid rafts and caveolae, as well as to junctional complexes and desmosomes/hemidesmosomes and to other constituents of the cytoskeleton, microtubules and intermediate filaments, thereby being able to read the condition prevalent in a cell. The actin filament system is linked to protein complexes at the cytoplasmic face of lipid rafts and caveolae, which arrangement enables the actin filaments to monitor the effectors' parts of the cell. Thereby the signals emitted by the actin filaments induce counteractions, enabling the cell to maintain its normal status. These actin linked proteins are e.g. galectin, filamin and flotillin, and they often form oligomers. The flotillin-1 and flotillin-2 form oligomers, mostly tetramers, which are anchoring lipid rafts to the actin filament network. Flotillin-1 binds with very high affinity to the AF protein as well as to the AF-derived peptide AF-16, while flotillin-2 is firmly linking actin filaments. The level of flotillin-1 is rapidly adjusted by degradation of free flotillin-1, rendering dynamics to the system. It is known that flotillins monitor certain kinases and phosphatases, which in turn regulate the activity of transmembrane proteins, e.g. ion pumps and G-protein linked systems such as NKCC's, the activity of which are further linked to the levels and activities of CAP/ponsin and FAK.
Optimization of drug and gene delivery is a topic of great interest. The optimization can be achieved via site-specific and targeted delivery, controlled drug release, and by finding ways to deliver higher concentrations of a drug into tissues of interest despite various barriers. Targeted delivery can serve to lower the required drug dose and minimize toxic side effects, which is e.g. crucial for the success of cancer treatment by immunotherapy, chemotherapy and/or radiation. Controlled release of drugs can be advantageous in the management of chronic diseases such as trauma-related conditions, neurodegenerative diseases, diabetes and hypertension.
Chemotherapy and immunotherapy are therapeutic approaches of major importance for the treatment of both localized and metastasized tumors. Since anticancer drugs are neither specific nor targeted to the cancer cells, improved delivery of anticancer drugs to tumor tissues in humans appears to be a reasonable, beneficial and achievable challenge. Scientists are working to increase the availability of drug for tumor uptake by 1) delaying the release preparations for long-lasting actions; 2) using liposome-entrapped drugs for prolonged effect or reduced toxicity; 3) administrating inert, non-toxic prodrugs for specific activation at the tumor site; 4) delivering antibody-mediated drugs; or 5) conjugating site-specific carriers to direct the drug to the tumor target. The latter depends heavily on pharmacokinetic investigations. Some success has been achieved in enhancing the efficacy and reducing the toxicity of drugs.
What is more, it is generally known in the field that the response of tumor to various anticancer drugs is tumor-size dependent in many aspects. In general, problems stem partly from the fact that the entire tumor cell populations do not respond equally to a certain treatment. As a result of recent progress in cancer biology, it has become evident that cellular heterogeneity of the tumor underlies the difficulties of treating primary and metastatic tumors with chemotherapy. Moreover, as tumors grow, marked diversity develops on the tissue level as well. An uneven distribution with an increase of areas of lower growth fraction and of poorer drug delivery is more distinct in larger tumors. Heterogeneous distribution and low levels of tumor blood flow are considered to be causally related to the heterogeneous nature of tumor tissue. Considering the lack of evidence of a lymphatic system within the tumor, increased interstitial fluid pressure may be a natural result that further impedes blood flow in the tumor.
The efficacy in cancer treatment of novel therapeutic agents such as monoclonal antibodies, cytokines and effector cells has been limited by their inability to reach their target in vivo in adequate quantities. Molecular and cellular biology of neoplastic cells alone has failed to explain the non-uniform uptake of these agents. This is not surprising, since a solid tumor in vivo is not just a collection of cancer cells. In fact, it consists of two extracellular compartments: vascular and interstitial. Since no blood-borne molecule or cell can reach cancer cells without passing through these compartments, the vascular and interstitial physiology of tumors has received considerable attention in recent years. Three physiological factors responsible for the poor localization of macromolecules in tumors have been identified: (i) heterogeneous blood supply, (ii) elevated interstitial pressure, and (iii) long transport distances. The first factor limits the delivery of blood-borne agents to well-perfused regions of a tumor; the second factor reduces extravasations of fluid, nutritients, oxygen and macromolecules in the high interstitial pressure regions and also leads to an experimentally verifiable, radial outward convection in the tumor periphery which opposes the inward diffusion. The third factor increases the time required for slowly moving macromolecules, nutritients, or oxygen to reach distant regions of a tumor. Binding of any molecule to e.g. an antigen further lowers the effective diffusion rate by reducing the concentration of mobile molecules. Although the effector cells are capable of active migration, peculiarities of the tumor vasculature and interstitium may also be responsible for poor delivery of lymphokine activated killer cells and tumor infiltrating immuno active cells in solid tumors. Due to micro- and macroscopic heterogeneities in tumors, the relative magnitude of each of these physiological barriers would vary from one location to another and from one day to the next in the same tumor, and from one tumor to another. If genetically engineered macromolecules and effector cells, as well as low molecular weight cytotoxic agents, are to fulfill their clinical promise, strategies must be developed to overcome or exploit these barriers.
Solid tumors, enclosed by a capsule and sometimes divided by septa, often develop high interstitial fluid pressure (IFP) as a result of increased fluid leakage and impaired blood circulation and lymphatic drainage, as well as changes in the extracellular matrix composition and elasticity. This means that the arteriolar blood pressure at many occasions causes the IFP to reach high levels. Also swelling of tumor cells contributes to the raised IFP. Raised interstitial fluid pressure forms a barrier to drug delivery and hence, resistance to therapy.
A cell undergoes genetic and epigenetic changes during its transition to malignancy. Malignant transformation is also accompanied by a progressive loss of tissue homeostasis and perturbations in tissue architecture that ultimately culminates in tumor cell invasion of the parenchyma and metastatic spread to distant tissue and organ sites. Increasingly, cancer biologists have begun to recognize that a critical component of this transformation journey involves marked alterations in the mechanical phenotype of the cell and its surrounding microenvironment. These include modifications in cell and tissue structure, adaptive force-induced changes in the environment, altered processing of micromechanical cues encoded in the extracellular matrix (ECM), and cell-directed remodeling of the extracellular stroma. Solid tumors are commonly stiffer than normal tissue, and tumors have altered integrins.
Growing evidence indicates that critical steps in cancer progression such as cell adhesion, migration, and cell cycle progression are in parts regulated by the composition and organization of the microenvironment. The adhesion of cancer cells to components of the microenvironment and the forces transmitted to the cells via the actin network and the signaling complexes organized at focal adhesions, lipid rafts and caveolae, allow cancer cells to sense the local topography of the extracellular matrix and respond efficiently to growth and migration promoting cues.
The cytoskeleton, including its actin network, is known to be of crucial importance for the structure and function of normal, inflammatory and neoplastic cells. At e.g. a brain trauma, the cytoskeleton is extensively deranged at locations and to an extent varying with the applied forces. The actin filaments are as well disintegrating at encephalitis (Jennische et al., 2008) and at cholera toxin induced diarrhea (Hansson et al., 1984). Thus, the crucial roles of the cytoskeleton for the maintenance of normal dimensions of cells and for the emergence of dysfunctions are established.
Much attention has focused on the role of membrane chloride (Cl−) channels in the maintenance of normal cell functions, emerging evidence highlights the importance of the Na+—K+2Cl− co-transporter (NKCC) as an independent regulatory site that may determine the overall rate of cell secretion. The co-transporter NKCC1 is expressed in virtually all mammalian cells, where it plays a more generalized role in cell volume homeostasis, cell ionic composition, and, possibly, the control of cell growth. Emerging molecular evidence indicates that NKCC1 function is regulated in the short and long term at the level of protein phosphorylation, membrane targeting, and gene expression (Mathews, 2002). Thus, an improved understanding of the interactions between the cytoskeleton, flotillin oligomers, lipid rafts and effectors such as NKCC1 has lead the present inventors to new therapeutic approaches to cancer and to neurodegeneration, as well as to the treatment of a range of clinical conditions in which the cell dimensions and ion composition are disturbed.
The Na—K—Cl co-transporters are a class of membrane proteins that transport Na+, K+, and Cl− ions into and out of a wide variety of epithelial and non-epithelial cells. The transport process mediated by Na—K—Cl co-transporters is characterized by electro neutrality (almost always with stochiometry of 1 Na:1K:2Cl) and inhibition by the “loop” diuretics such as bumetanide, benzmetanide, and furosemide. Presently, two distinct Na—K—Cl co-transporter isoforms have been identified by cDNA cloning and expression; genes encoding these two isoforms are located on different chromosomes and their gene products share approximately 60% amino acid sequence identity.
The NKCC1 (CCC1, BSC2) isoform is present in a wide variety of tissues. Most normal epithelial cells containing NKCC1 are secretory epithelia with the Na—K—Cl co-transporter localized to the basolateral membrane. By contrast, NKCC2 (CCC2, BSC1) is found only in the kidney, localized to the apical membrane of the epithelial cells of the thick ascending limb of Henle's loop and of the macula densa. Mutations in the NKCC2 gene result in Bartter's syndrome, an inherited disease characterized by hypo potassium metabolic alkalosis, hypercalciuria, salt wasting, and volume depletion. The two Na—K—Cl co-transporter isoforms are also part of a superfamily of cation-chloride co-transporters, which includes electroneutral K—Cl and Na—Cl co-transporters. Cancer cells, which mostly are apolar, do express high levels of NKCC, resulting in that the tumor cells in fact are swollen, i.e. having increased dimensions. Tumor cells have less precisely regulated ion pump and water channel systems, but still show a strong tendency to maintain their internal homeostasis. That means that the increased dimensions of tumor cells, enclosed by a capsule, contribute to the raise of the IFP common in solid tumors.
Na—K—Cl co-transporter activity is affected by a large variety of hormonal stimuli as well as by changes in cell volume. In many tissues this regulation (particularly of the NKCC1 isoform) is regulated by the balance between phosphorylation and dephosphorylation of regulatory systems, controlling the ion pumps prevalent in the lipid rafts through the specific protein kinases or phosphatases. (Haas, 1998)
Cell shrinkage-induced activation of NKCC involves an interaction between the cytoskeleton and protein phosphorylation events via PKC and myosin light chain kinase (MLCK). Osmotic control of Cl— secretion across the epithelium includes: (i) hyperosmotic shrinkage activation of NKCC1 via PKC, MLCK, p38, OSR1 and SPAK; (ii) deactivation of NKCC by hypotonic cell swelling and a protein phosphatase, and (iii) a protein tyrosine kinase acting on the focal adhesion kinase (FAK) to set levels of NKCC activity. The CAP component is interposed as well and takes parts in the step wise regulation of the extent of phosphorylation of NKCC, which determines its function within the lipid rafts (Hoffmann 2007).
At the electron microscopic level, a unique combination of integrin β1, the phosphorylated form of FAK at tyrosine 407 (pY407) and Na(+), K(+), 2Cl(−) co-transporter (NKCC1) were all co-localized only on the basolateral membrane in normal cells. The three proteins were also co-immunoprecipitated with each other in isotonic conditions, suggesting an osmosensing complex involving the three proteins. Only FAK pY407 was sensitive to hypotonic shock and became dephosphorylated with hypotonic shock, while FAK pY576 in the apical membrane and pY861 in cell-cell adhesions were insensitive to hypotonicity. It has been reported that chloride cells respond to hypotonic shock using integrin β1 as an osmosensor that is connected to dephosphorylation of FAK pY407 which leads to NKCC1 deactivation in the basolateral membrane and the inhibition of NaCl secretion by these epithelial cells (Marshall, 2008). Again, tumor cells commonly are apolar and thus the ion pumps in the lipid rafts are localized all along the cell surface, as apical and basolateral areas are not prevalent. Thus, the same kind of ion pump is prevalent in normal cells as in cancer cells and in either case similarly monitored, albeit their localizations differ.
Integrins are cell surface receptors which, in part, mediate the adhesion of cells to the extracellular matrix. In addition to providing molecular “glue” essential for tissue organization and survival, integrins serve as dynamic signaling molecules. Integrins allow normal, non-transformed cells to sense that they are adhered to the extracellular matrix, thus providing a cell survival signal. This signal allows cells to proliferate in the presence of growth factors and in some instances prevents apoptosis. Integrins also mediate cell migration as it occurs in normal processes, such as angiogenesis, wound healing, repair of damage, monitor immune system function, and development. Aberrances in the expression and function of integrins contribute to many diseases and disorders, including cancer.
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in a variety of cancers and plays an important role in cell adhesion, migration, and anchorage-dependent growth (Tilgham, 2007)
Focal adhesion kinase (FAK), prevalent in practically all normal cells, is overexpressed in invasive and metastatic colon, breast, thyroid, and prostate cancers. Enhanced FAK immunostaining is detected in small populations of preinvasive (carcinoma in situ) oral cancers and in large populations of cells in invasive oral cancers. It has been hypothesized that FAK is probably not a classical oncogene but may be involved in the progression of cancer to invasion and metastasis. It is further hypothesized that overexpression of FAK in subpopulations of tumor cells leads to populations of cells with a high propensity toward invasion and metastasis (Kornberg, 1998).
Focal adhesion kinase (FAK) localizes to cellular focal adhesions or cell contacts within the extracellular matrix. FAK is activated by a variety of cell surface receptors and transmits signals to a range of targets. FAK participates in growth factor receptor-mediated signaling pathways and plays essential roles in cell survival, proliferation, migration, and invasion.
Overexpression of FAK is widely observed in numerous tumor types, and is used as a marker for invasion and metastasis. FAK could be therapeutically targeted at various levels, such as at the level of FAK gene transcription by regulating its transcription factor(s) with siRNA, at the FAK mRNA level with FAK siRNA, or at the protein level. At the protein level, FAK's localization to lipid rafts in focal adhesions could be disrupted by expression of dominant-negative FAK-Related Non-Kinase or its focal adhesion targeting domain, and its kinase activity could be inhibited by FIP200, the FAK kinase domain-interacting protein and kinase-activity inhibitor. In recent years, research has been focused on developing small molecule inhibitors against FAK transcription and activation, to provide additional approaches for potential tumor therapies (Lis. 2008).
Another substrate involved in the phosphorylation of intracellular substrates regulating the transduction and control of signals determining the level of activity of ion pumps is CAP, which is an adapter protein for the Cbl proto-oncogene product. CAP is acting as a link in the signaling pathway at the cytoplasmic leaflet of lipid rafts between flotillin and FAK. CAP is also known under the name ponsin. The name reflects that this factor originally was isolated and identified in over-expressing tumors and therefore named proto-oncogene product, but has subsequently been disclosed to be prevalent as well in normal cells.
Antisecretory factor is a 41 kDa protein that originally was described to provide protection against diarrhea diseases and intestinal inflammation (for a review, see Lange and Lönnroth, 2001). The antisecretory factor (AF) protein has been sequenced and its cDNA cloned. The antisecretory activity seems to be mainly exerted by a peptide located between the amino acid positions 35 and 50 on the antisecretory factor (AF) protein sequence and comprising at least 4-16, such as 4, 6, 8 or 16 amino acids of the consensus sequence. Immunochemical and immunohistochemical investigations have revealed that the antisecretory factor (AF) protein is present and may also be synthesized by most tissues and organs in a body. Synthetic peptides, comprising the antidiarrhoeic sequence, have prior been characterized (WO 97/08202; WO 05/030246). Antisecretory factor (AF) proteins and peptides have previously been disclosed to normalize pathological fluid transport and/or inflammatory reactions, such as in the intestine and the choroid plexus in the central nervous system after challenge with the cholera toxin (WO 97/08202). Food and feed with the capacity to either induce endogenous synthesis of AF or uptake of added AF have therefore been suggested to be useful for the treatment of edema, diarrhea, dehydration and inflammation in WO 97/08202. WO 98/21978 discloses the use of products having enzymatic activity for the production of a food that induces the formation of antisecretory factor (AF) proteins. WO 00/038535 further discloses the food products enriched in antisecretory factor (AF) proteins as such.
Antisecretory factor (AF) proteins and fragments thereof have also been shown to improve the repair of nervous tissue, and proliferation, apoptosis, differentiation, and/or migration of stem and progenitor cells and cells derived thereof in the treatment of conditions associated with loss and/or gain of cells (WO 05/030246) and to be equally effective in the treatment and/or prevention of intraocular hypertension (WO 07/126,364), as for the treatment and/or prevention of compartment syndrome (WO 07/126,363).
What is more, the present inventors recently showed that antisecretory factors were able to monitor and/or beneficially affect the structure, distribution and multiple functions of lipid rafts, receptors and/or caveolae in membranes and could thus be employed for the treatment and/or prevention of structural disorganization and dysfunction of lipid rafts and/or caveolae in cell membranes (WO 07/126,365).
Surprisingly, the present inventors have now been able to prove that the same antisecretory factors can intervene in the above described biological activation of transmembrane proteins, e.g. NKCC1 through FAK and CAP, and can thus directly regulate the pathological activity of the ion channel in pathological and/or perturbed cells, effectively normalizing the intracellular pressure and transmembrane protein function in said cell, and thus allowing an improved uptake of drugs used in e.g. cancer therapy.